Calcitonin salmon analogues

ABSTRACT

Salcatonin (i.e. salmon calcitonin) analogues of formula R1-Ser-Asn-Leu-Ser-Thr-Cys(SR2)-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 (I) are disclosed, in which R1=-Cys-S-H, -Cys-OH, -Cys-S-ether or -Cys-S-ester (where the ether or ester residue has 2-5C) or a corresponding salt or isomer; R2=H, OH, ester or ether residue of 2-5C or acetamidomethyl, or a corresponding salt or isomer.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of PCT Application Ser. No.PCT/EP96/00487, filed Feb. 2, 1996, designating the United States ofAmerica.

This invention relates to novel substituted salmon calcitonin(salcatonin) analogues. More particularly this invention relates tosalcatonin analogues, which are useful for the prevention or treatmentof diseases such as osteoporosis, hypercalcemia, Paget's disease, asreference for analytical testing, and to methods of production and usethereof.

Salcatonin and its preparations, having hypocalcemic properties, aredisclosed in the following documents:

    ______________________________________                                        GB Pat. No. 82-28390 Oct. 05, 1982                                            GB Pat. No. 84-7907        Mar. 27, 1984                                      JP Pat. No. 63316737      Dec. 26, 1988                                       EP Pat. No. 302772          Feb. 08, 1989                                     EP Pat. No. 327756          Aug. 16, 1989                                     JP Pat. No. 1230530        Sept. 14, 1989                                     EP Pat. No. 364235          Apr. 18, 1990                                     JP Pat. No. 03052821      Mar. 07, 1991                                       EP Pat. No. 91109497      June 10, 1991                                       EP Pat. No. 471618          Feb. 19, 1992                                     WO Pat. No. 9306854        Apr. 15, 1993                                      WO Pat. No. 9408622        Apr. 28, 1994                                      ______________________________________                                    

The prior art documents are all concerned with calcitonin salmon(salcatonin) raw material and its preparations, having hypocalcemicproperties.

The calcitonin salmon molecule is characterized by a S--S bridge betweenthe Cysteine in position 1 and the Cysteine in position 7. Theapplicants for the present invention have surprisingly found thatanalogues of calcitonin salmon molecule having the disulphide bridgebetween the positions 1 and 7 broken, with the possibility ofintroduction of various substituents confers significant advantages interms of hypocalcemic activity and use as analytical agents.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: A graph illustrating the age of the tested salcatoninpreparation in the range of 24 months.

The novel salcatonin analogues of this invention, useful as hypocalcemicagents, substituted as 31 or 32 amino acids, are represented by thefollowing formula: ##STR1## wherein R₁ is -Cys-SH or Cys-S-OH: or-Cys-S-ether or -Cys-S-ester wherein the ether or ester has from 2 to 5carbon atoms or pharmaceutically acceptable salts or isomers thereof; R₂is --H, --OH, an ester or ether having from 2 to 5 carbon atoms or,

pharmaceutically acceptable salts or isomers thereof.

Preferably R₁ and R₂ are as follows:

    ______________________________________                                         COMPOUNDS  R.sub.1        R.sub.2                                            ______________________________________                                        Compound 1  --Cys--SH      --OH                                               Compound 2      --Cys--S--OH                                                                                               --H                              Compound 3      --Cys--SH                 --OOC--CH.sub.3                     Compound 4      --Cys--SH                 --OOC--C.sub.2 H.sub.5              Compound 5      --Cys--SH                 --OOC--C.sub.3 H.sub.7              Compound 6      --Cys--SH                 --OOC--C.sub.4 H.sub.9              Compound 7      --Cys--S--OOC--CH.sub.3                                                                              --H                                    Compound 8      --Cys--S--OOC--C.sub.2 H.sub.5                                                              --H                                             Compound 9      --Cys--S--OOC--C.sub.3 H.sub.7                                                                      --H                                     Compound 10    --Cys--S--OOC--C.sub.4 H.sub.9                                                               --H                                             Compound 11    --Cys--SH                  --O--C.sub.2 H.sub.5                Compound 12    --Cys--SH                  --O--C.sub.3 H.sub.7                Compound 13    --Cys--SH                  --O--C.sub.4 H.sub.9                Compound 14    --Cys--SH                  --O--C.sub.5 H.sub.11               Compound 15    --Cys--S--O--C.sub.2 H.sub.5                                                                    --H                                          Compound 16    --Cys--S--O--C.sub.3 H.sub.7                                                                   --H                                           Compound 17    --Cys--S--O--C.sub.4 H.sub.9                                                                    --H                                          Compound 18    --Cys--S--O--C.sub.5 H.sub. 11                                                                --H                                            ______________________________________                                    

Compounds 1 and 2 or their mixture are also conventionally definedhereinafter as hydroxysalcatonins.

Preferred embodiments of the present invention are

(i) when R₁ is --Cys--S--OH or an ester R₂ is a hydrogen atom, or

(ii) when R₂ is ═-OH or an ester, R₁ is -Cys-H.

This invention also includes pharmaceutically acceptable salts of anysuitable inorganic or organic acids. Suitable inorganic acids are, forexample, hydrochloric, hydrobromic, sulfuric and phosphoric acids.

Suitable organic acids include carboxylic acids such as acetic,propionic, glycolic, lactic, pyruvic, malonic, succinic, fumaric, malic,tartaric, citric, ascorbic and maleic acids.

The above salts may be prepared by conventional means, already wellknown to a person skilled in the art.

It has now been surprisingly discovered that the compounds of thisinvention are useful as hypocalcemic agents, inhibiting physiological orpathological bone re-absorption.

In fact the inhibition of bone re-absorption may be determined by adecrease of urinary excretion of hydroxyproline and that associated withthe reduction of high and pathological serum levels of alkalinephosphatase and with the normalization of calcium balance, promotescollagen and bone tissue rebuilding, reducing the total blood content ofCa²⁺ after some months of treatment.

Moreover salcatonin analogues exert antalgical action, reducingpartially or entirely the pain.

Salcatonin analogues may be used in Paget's disease (osteitisdeformans), hypercalcaemia caused by cancers, hyperparathyroidism,intoxication of Vit. D, both as emergency treatment and prolonged aswell, osteoporosis of different origin, optionally combined with otherassociated therapy, specific for each unhealthy condition and Sudeck'sdisease.

In order to evaluate the pharmacological activity of salcatoninanalogues, a preliminary screening by biological assay was carried out,as indicated in BP (British Pharmacopoeia) 1993 Volume I, page 587,using three different dosages (8 mI.U./rat, 16 mI.U/rat, 32 mI.U./rat),obtaining the following results, reported hereby, expressed as averagevalue of three determinations:

    ______________________________________                                                                   AVERAGE                                                                       VALUE                                              COMPOUND                   (mg Ca %)                                          ______________________________________                                        Compound 1                                                                             (R.sub.1 =  Cys--SH; R.sub.2 =  --OH)                                                               4.62                                           Compound 2                                                                               (R.sub.1 =  Cys--S--OH; R.sub.2 =  --H)                                                                         4.48                             Compound 3                                                                               (R.sub.1 =  Cys--SH; R.sub.2 =  --OOC--CH.sub.3)                                                            2.65                                 Compound 4                                                                               (R.sub.1 =  Cys--SH; R.sub.2 =  --OOC--C.sub.2 H.sub.5)                                                    2.83                                  Compound 5                                                                               (R.sub.1 =  Cys--SH; R.sub.2 =  --OOC--C.sub.3 H.sub.7)                                                    2.51                                  Compound 6                                                                               (R.sub.1 =  Cys--SH; R.sub.2 =  --OOC--C.sub.4 H.sub.9)                                                    2.19                                  Compound 7                                                                               (R.sub.1 =  Cys--S--OOC--CH.sub.3 ; R.sub.2 =                                                             2.32                                   Compound 8                                                                               (R.sub.1 =  Cys--S--OOC--C.sub.2 H.sub.5 ; R.sub.2 =                                                       2.56                                  Compound 9                                                                               (R.sub.1 =  Cys--S--OOC--C.sub.3 H.sub.7 ; R.sub.2 =                                                      2.78                                   Compound 10                                                                             (R.sub.1 =  Cys--S--OOC--C.sub.4 H.sub.9 ; R.sub.2 =                                                       2.07                                   Compound 11                                                                             (R.sub.1 =  Cys--SH; R.sub.2 =  --O--C.sub.2 H.sub.5)                                                        2.87                                 Compound 12                                                                             (R.sub.1 =  Cys--SH; R.sub.2 =  --O--C.sub.3 H.sub.7)                                                        2.62                                 Compound 13                                                                             (R.sub.1 =  Cys--SH; R.sub.2 =  --O--C.sub.4 H.sub.9)                                                        2.57                                 Compound 14                                                                             (R.sub.1 =  Cys--SH; R.sub.2 =  --O--C.sub.5 H.sub.11)                                                      2.74                                  Compound 15                                                                             (R.sub.1 =  Cys--S--O--C.sub.2 H.sub.5 ; R.sub.2 =                                                           2.91                                 Compound 16                                                                             (R.sub.1 =  Cys--S--O--C.sub.3 H.sub.7 ; R.sub.2 =                                                           2.85                                 Compound 17                                                                            (R.sub.1 =  Cys--S--O--C.sub.4 H.sub.9 ; R.sub.2 =                                                           2.23                                  Compound 18                                                                            (R.sub.1 =  Cys--S--O--C.sub.5 H.sub.11 ; R.sub.2 =                                                         2.46                                   ______________________________________                                    

The compounds of the present invention are advantageously formulated inliquid forms such as solutions or suspensions administrable forparenteral route, nasal spray or as rectal capsules.

The injectable dosage forms are solutions or suspensions of the abovecompounds in a suitable pharmaceutical carrier, which is a sterileliquid such as water with or without the addition of otherpharmaceutically acceptable excipients or adjuvants.

For use as nasal spray, the solution or suspension of salcatoninanalogues of this invention may be formulated as aqueous preparationswith or without convenient excipients, packed in suitable containersequipped with pressurized inhalers or nebulizing or atomizing--dosingpumps.

The recommended daily dose of novel compounds described herein mayquantitatively vary over a wide range of from 10 I.U. to 400 I.U., morepreferably from 100 I.U. to 200 I.U., in order to provide in a unitdosage an effective amount of active analogue.

As used herein the term "I.U." refers to the appropriate InternationalReference Preparation (I.R.P.) of human, salmon, porcine, eelcalcitonins established by the National Institute for BiologicalStandards and Control, Blanche Lane, South Mimms, Potters Bar,Hertfordshire, E6 30G, U.K.

Another preferred embodiment of the present invention is that the novelcompounds, more specifically hydroxysalcatonins, may be advantageouslyused as selective and practical indicators or markers to determine theage and consequently the shelf-life of salcatonin preparations.

In fact it was also surprisingly discovered that salcatonin solutions,due to their instability in the presence of oxygen (O₂) at roomtemperature, progressively degrade producing appreciable quantities ofhydroxysalcatonins.

When the solutions are exposed to O₂, the degradation process is sofast, that remarkably variable (inconstant) quantities ofhydroxysalcatonins are produced, so that it is difficult to establishany relationship between hydroxysalcatonins content and the age of thesalcatonin preparation.

For these reasons it is preferred that manufacture of salcatoninsolutions is carried out under saturated nitrogen atmosphere (about1×10⁵ Pa (1 bar)), in order to reduce the degradation process. It isalso preferred the material is stored under refrigerated temperatureconditions.

In order to establish the linearity of the degradation process involvingsalcatonin preparations, analytical test conditions have beenstandardized and experimental samples have been prepared with an oxygencontent ≦1 p.p.m. and stored separately either at temperatures of from4° to 8° C. or at 22° C.

Under these conditions it has been unexpectedly demonstrated that it ispossible to determine the age and consequently the shelf-life of asalcatonin preparation once the quantity of hydroxysalcatonins, producedduring the storage period, is analytically determined, using thesynthetized hydroxysalcatonins of the invention as reference standards.

In fact the enclosed Graph 1, shows analysis of the content ofhydroxysalcatonins at different intervals on six batches of salcatoninpreparations (ampoules 100 I.U./ml), demonstrates a good linearitybetween the age of the formulations and the above reference standard,thus representing a practical and suitable parameter for the assessmentof their shelf-life.

Hydroxysalcatonins are synthetized from salcatonin (batch No. 6Q1,branded UCB-Bioproducts, Belgium) by an oxidation process using suitableoxidizing agents, such as H₂ O₂, performic acid, percloric acid, O₃,chloranil or an equivalent thereof, to open the disulphur bridge betweencysteines 1-7 of salcatonin and then oxidize preferably cysteine inposition 1, while small quantities of oxidized cysteine in position 7may also be obtained.

The first step is carried out in a flask, where salcatonin is completelydissolved in suitable dilute acids and anhydrous methanol is added withstirring, in order to prevent freezing of the solution during oxidation.

The salcatonin solution and the oxidizing agent are transferred to theseparate arms of a glass stoppered test-tube to which a side arm hasbeen sealed.

The reagents are cooled during 30 minutes in a bath maintained from -7°C. and -10° C. (or at 0° C.) and then mixed by tipping the tube.

The reaction is allowed to take place at the same controlled temperaturefor approximately 2.5 hours.

The obtained hydroxysalcatonins are then purified on a conventionalseparatory column containing a suitable resin (Spherisorb™ C₁₈, 250×4.6mm, 5μ) and, once the solution has been chromatographed andhydroxysalcatonins separated from the mixture of salcatonin and otherpotential related substances, the synthetized analogues are mobilizedfrom the corresponding portion of the resin by using acetonitrile, as asolvent.

Acetonitrile is then completely removed from the solution containinghydroxycalcitonins by using conventional methods.

In order to use hydroxysalcatonins as a reference standard foranalytical tests, stock solutions of the same in water are prepared,kept frozen at about -80° C., lyophilized and used no longer than 6months from their preparation.

Just before use, the lyophilized hydroxysalcatonins are admixed to thesame suitable vehicle as that of the sample to analyze, in order toobtain an adequate reference standard solution.

This standard at known concentration is then injected in an HPLC/MSsystem, thus obtaining calibration graphs, which are later on used toidentify the peaks and to determine quantitatively thehydroxysalcatonins in the stored salcatonin solutions, in order toassess the exact age of the tested preparations by comparing them withthe standard graph.

Analytical determinations are carried out with a Perkin Elmer MassSpectrometer Sciex API III equipped with an "Ion-spray" ionizationssource.

The mass Spectrometer is connected to an Applied Biosystems 140A syringeHPLC with a Perkin Elmer ISS-101 autosampler.

The column is eluted with a gradient indicated hereby:

(A) acetonitrile+0.1% trifluoroacetic acid (TFA)

(B) water+0.1% TFA.

The microbore column is eluted at 50 μl/min.

The gradient begins with 10' in isocratic conditions at 100% B followedby a first linear step that takes to an 80% of B in 5' and by a secondlinear step that reaches the 50 % of B in 20'; this condition is thenisocratically maintained for 15'. For the analysis on microbore columnthe different samples of salcatonin preparations under testing areinjected without extraction or clean-up steps; the volume of eachinjection is 500 μl.

For the reference standard the injections are of the same volume fromsolutions at 100 ng/ml prepared with lyophilized product dissolved inthe same solvent mixture of the samples, just before use.

HPLC-MS analyses are carried out on-line during chromatographicseparations on microbore column without splitting.

MS analyses are performed in scanning mode, range from m/z 400 to 2000,acquiring positive ions.

Some samples are then analyzed with MS/MS techniques using argon ascollision gas (collision gas thickness 3×10⁴ atoms/cm³) with a collisionenergy of from 50 to 200 eV.

In order to obtain quantitative determinations of hydroxysalcatoninswith higher sensitivity and accuracy, the instrument is then operated inselected ion monitoring, choosing m/z values characteristic of the aboveproducts.

The results of the above analytical test (see Graph 1) demonstrate thathydroxysalcatonins are essential and suitable indicators to evaluate theage of salcatonin solutions, once they are prepared in nitrogenatmosphere and stored under controlled temperature conditions.

In fact the above results evidence a clear linearity between the contentof hydroxysalcatonin(s) and the age of the analyzed samples.

The invention is further illustrated by the following examples, but inany case they are given by way of illustration only and are not to beconstrued as limiting the invention either in spirit or in scope as manymodifications will be apparent to those skilled in the art.

EXAMPLE 1

Synthesis process of hydroxysalcatonins.

200 mg of salcatonin (batch No. 6Q1, UCB-Bioproducts, Belgium) wascompletely dissolved in 5.0 ml of formic acid (99%) and then 1.0 ml ofanhydrous methanol was added.

Performic acid was prepared separately by adding 0.50 ml of 30% H₂ O₂ to9.5 ml of 99% of formic acid and the resulting solution was placed atroom temperature (25° C.) for 2 hours in a stoppered flask.

The salcatonin solution and the performic acid were transferred toseparate arms of a glass-stoppered test-tube to which a side arm issealed.

Both reagents were cooled for approximately 30 minutes in a bathmaintained at a temperature of from -7° C. to 10° C. and then mixedtogether by tipping the tube.

The reaction took place at a steady temperature for approximately 2.5hours.

The obtained hydroxysalcatonins were purified using the followingpreparative method:

    ______________________________________                                         Equipment:   5060B Varian HPLC connected to a Gilson                                         pump (Model 302) for the injection of the                                                  sample.                                          Column:        Spherisorb ™  C.sub.18  (250 ×  4.6 mm, 5μ)        Eluent:        Gradient formed by:                                                          A)                                                                            acetonitrile +  0.1% trifluoroacetic acid                                                      (TFA)                                                    B)  water +  0.1% TFA                                                Flow rate:  1 ml/min                                                         ______________________________________                                    

The hydroxysalcatonins were mobilized from the relevant portion of thecolumn by using acetonitrile, which was then completely removed from thesolution by conventional methods already well known in the art. A yieldof 72.7% was obtained.

EXAMPLE 2

Preparation of 5,000 ampoules of hydroxysalcatonins (0.02 mg/ml). 1 mlof solution containing:

    ______________________________________                                         Hydroxysalcatonins    0.02   mg                                              Glacial acetic acid               mg   2.00                                   Sodium acetate trihydrate                                                                                       mg0                                         Sodium chloride                   mg      7.50                                Water for injections              mg  1.00                                    ______________________________________                                    

Approximately 4.5 liters of water, for injections was introduced into astainless steel dissolutor, previously sterilized and depyrogenated bysteam and then cooled at about 8° C. The dissolutor was equipped with astirrer and hermetically sealed under controlled bacteriologicalconditions by introducing filtered sterile nitrogen.

Sodium acetate trihydrate and glacial acetic acid were added underconstant and slow stirring until complete dissolution of theingredients.

The pH of the buffer solution was measured to be in the range of4.2±0.3.

The total quantity of hydroxysalcatonins were dissolved separately in a25 ml sterile and apyrogen Erlenmayer flask by introducing about 5 ml ofcold buffer solution, thus obtaining the mother solution ofhydroxysalcatonins.

Once the hydroxysalcatonins were completely dissolved the mothersolution of hydroxysalcatonins, was added under constant and slowstirring to the base solution.

The remaining water for injectable preparations was added to thedissolutor to yield 5 Kg of solution and the pH was checked again (to bebetween pH 4.2±0.3).

The obtained solution was then sterilized by filtration, using apre-cartridge 0.45 μm pore size (brand PALL), followed by a secondcartridge 0.22 μm pore size (brand PALL) and finally was automaticallydivided in ampoules each containing 1 ml.

EXAMPLE 3

Preparation of 2,000 nasal spray bottles. (0.9 ml; 0.02 mg/ml) ofhydroxysalcatonins.

1 ml of the solution containing:

    ______________________________________                                        Hydroxysalcatonins     0.02    mg                                             Sodium citrate dihydrate                                                                                     mg12.36                                        Citric acid rnonohydrate                                                                                     mg12.11                                        Sodium taurocolate             mg      10.00                                  Disodium edetate               mg         1.00                                Benzalkonium chloride          mg    0.23                                     Distilled water                mg          1.00                               ______________________________________                                    

1.0 kg of distilled water was placed in a sterilized stainless steelcontainer hermetically sealed, under controlled bacteriologicalconditions by introducing filtered sterile nitrogen.

A solution (A) was prepared separately in about 0.5 liter of distilledwater to contain:

    ______________________________________                                        Sodium citrate dihydrate                                                                             24.72  g                                               Citric acid monohydrate                                                                                     g   24.22                                       Sodium taurocolate            g        20.0                                   Disodium edetate              g           2.0                                 ______________________________________                                    

Once the above solution had been completely dissolved, the pH value waschecked to be in the range of from 3.6 to 4.5 and it was added to thewater into the dissolutor.

Under constant and slow stirring 0.46 g of benzalkonium chloride wasadded.

The total quantity of hydroxysalcatonins was dissolved separately in 50ml of solution (A) in an volumetric flask, thus obtaining the mothersolution of hydroxysalcatonins.

Once the hydroxysalcatonins were completely dissolved, the mothersolution was added to the dissolutor under constant and slow stirringand the remaining water (about 0.5 liter) was introduced until to yield2 liters of volume.

The obtained solution was filtered through a sterilizing filter (brandPALL, 0.22μ) and closed by the automatic filling operation into nasalspray bottles containing each 0.9 ml of solution.

EXAMPLE 4

Preparation of 4,000 nasal spray bottles (1.3 ml, 0.07 mg/ml) ofcompound 12. 1 ml of solution containing:

    ______________________________________                                        Compound 12            0.07   mg                                              Sodium chloride                     mg6.00                                    Sodium citrate dihydrate                                                                                   4.63                                                                               mg                                          Citric acid monohydrate                                                                                    3.00                                                                               mg                                          Methyl p-hydroxybenzoate                                                                                   1.30                                                                               mg                                          Propyl p-hydroxybenzoate                                                                                   0.20                                                                                  mg                                       Sodium acetate                      mg 1.00                                   Alkylamidopropylbetain              mg                                        Bidistilled water                 ml1.00                                      ______________________________________                                    

The bidistilled water (2.6 liters) was placed in a sterilized stainlesssteel container hermetically sealed, introducing filtered sterilenitrogen, in order to maintain the controlled bacteriologicalconditions.

A solution (A) was prepared separately in about 1.3 liters ofbidistilled water to contain:

    ______________________________________                                        Sodium chloride        31.20  g                                               Sodium citrate dihydrate                                                                                  24.07                                                                               g                                           Citric acid monohydrate                                                                                    15.60                                                                              g                                           Sodium acetate                    g      5.20                                 ______________________________________                                    

After the preparation of the above solution, the pH was checked to be inthe range of from 4.2±0.5 and it was added to the water into thedissolutor.

Under constant and slow stiring 6.76 g of methyl p-hydroxybenzoate, 1.04g of propyl p-hydroxybenzoate and 1.04 g of alkylamidopropylbetain wereadded.

Compound 12 (364 mg) was dissolved separately in 455 ml of solution (A)in a volumetric flask, thus obtaining the mother solution of compound12, which was added to the dissolutor under constant and slow stirring.

The remaining water (about 845 ml) was introduced to yield 5.2 liters ofsolution.

The resulting solution was filtered through a sterilizing filter (brandPALL, 0.22 μm) and dispensed into nasal spray bottles containing each1.3 ml of solution, by using conventional automatic filling.

While only certain embodiments of our invention have been described inspecific detail, it will be apparent to those skilled in the art thatmany other specific embodiments may be practised and many changes may bemade all within the spirit of the invention and the scope of theappended claims.

EXAMPLE 5

Chromatographic analytical determination of the age of a salcatoninpreparation (stored at 4°-8° C. temperature), using synthetichydroxysalcatonins as indicator of the degradation process.

Prepare a reference standard solution of pure hydroxysalcatonins havinga concentration of 10 ng/ml, by dissolving in 10 ml of distilled water100 ng of lyophilized hydroxysalcatonins, already synthetized asindicated in Example 1.

Use the salcatonin preparation under testing as it is, withoutextraction or clean-up steps.

The utilized HPLC/MS system is the following:

    ______________________________________                                        HPLC                                                                          Equipment:            Applied Biosystems 140A syringe HPLC                                         with a Perkin Imer ISS--101 autosampler                  Eluent:              (A)  acetonitrile +  0.1%                                               trifluoroacetic acid                                                          (B) water +  0.1% trifluoroacetic acid                         Flow rate:            50 μl/min.                                           Injection volume                                                                               500 μl                                                    MS                                                                            Equipment:           Perkin Elmer Mass Spectrofotometer                                        Sciex API III equipped with an on-                                            spray ionization source.                                     m/Z range:            400-2000 m/z                                            Collision gas:                                                                                    argon                                                     Thickness                                                                     collision gas:                                                                                  10.sup.4  atoms/cm.sup.3                                    Collision energy                                                                               50-200 eV.                                                   ______________________________________                                    

From the HPLC/MS study, the tested salcatonin preparation presented atotal hydroxysalcatonins content of 1.46%, compared with the referencestandard.

Using the above figure of hydroxysalcatonins total content as anindicator for the degradation processes, it is possible to determine onthe calibration Graph 1 the age of the tested salcatonin preparation inthe range of 12 months.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 1                                               - <210> SEQ ID NO 1                                                           <211> LENGTH: 32                                                              <212> TYPE: PRT                                                               <213> ORGANISM: salmon                                                        <220> FEATURE:                                                                <221> NAME/KEY: VARIANT                                                       <222> LOCATION: (1)                                                           #a modified Cys residue: position 1 may encompass                             - <400> SEQUENCE: 1                                                           - Xaa Ser Asn Leu Ser Thr Cys Val Leu Gly Ly - #s Leu Ser Gln Glu Leu         #                 15                                                          - His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Th - #r Gly Ser Gly Thr Pro         #             30                                                              __________________________________________________________________________

What is claimed is:
 1. A salcatonin analogue of the formula: ##STR2##wherein R₁ is -Cys-S-H or -Cys-S-OH or -Cys-S-ester or Cys-S-etherwherein the ester or ether has from 2 to 5 carbon atoms orpharmaceutically acceptable salts or isomers thereof andR₂ is hydrogenor hydroxy or an ester or an ether having from 2 to 5 carbons atoms or apharmaceutically acceptable salt of isomer thereof, provided that whenR₁ is -Cys-S--H, then R₂ is not hydrogen.
 2. A salcatonin analogue ofthe formula: ##STR3## Where R₁ and R₂ are selected from the following:

    ______________________________________                                             COMPOUNDS                                                                            R.sub.1         R.sub.2                                           ______________________________________                                        Compound 1  --Cys--SH       --OH                                              Compound 2             --Cys--S--OH                                                                                       --H                               Compound 3             --Cys--SH                                                                                        --OOC--CH.sub.3                     Compound 4             --Cys--SH                                                                                        --OOC--C.sub.2 H.sub.5              Compound 5             --Cys--SH                                                                                        --OOC--C.sub.3 H.sub.7              Compound 6             --Cys--SH                                                                                        --OOC--C.sub.4 H.sub.9              Compound 7             --Cys--S--OOC--CH.sub.3                                                                       --H                                    Compound 8             --Cys--S--OOC--C.sub.2 H.sub.5                                                               --H                                     Compound 9             --Cys--S--OOC--C.sub.3 H.sub.7                                                        --H                                            Compound 10           --Cys--S--OOC--C.sub.4 H.sub.9                                                        --H                                             Compound 11           --Cys--SH                                                                                         --O--C.sub.2 H.sub.5                Compound 12           --Cys--SH                                                                                             --O--C.sub.3 H.sub.7            Compound 13           --Cys--SH                                                                                             --O--C.sub.4 H.sub.9            Compound 14           --Cys--SH                                                                                             --O--C.sub.5 H.sub.11           Compound 15           --Cys--S--O--C.sub.2 H.sub.5                                                                    --H                                   Compound 16           --Cys--S--O--C.sub.3 H.sub.7                                                            --H                                           Compound 17           --Cys--S--O--C.sub.4 H.sub.9                                                            --H                                           Compound 18           --Cys--S--O--C.sub.5 H.sub.11                                                                  --H.                                   ______________________________________                                    


3. The salcatonin analogue of claim 1, wherein R₁ is -Cys-S--OH or-Cys-S-ester, wherein the ester has from 2 to 5 carbon atoms orpharmaceutically acceptable salts or isomers thereof, andR₂ is hydrogen.4. The salcatonin analogue of claim 1, whereinR₁ is -Cys-S--H, and R₂ ishydroxy or an ester having from 2 to 5 carbons atoms or apharmaceutically acceptable salt of isomer thereof.
 5. A pharmaceuticalformula comprising an analogue as claimed in claim
 1. 6. Apharmaceutical formula as claimed in claim 5 wherein the analogue is insolution or suspension in a suitable pharmaceutical carrier.
 7. Apharmaceutical formula as claimed in claim 6 further comprising sterilewater, other sterile liquid, pharmaceutically acceptable excipients oradjuvants or a combination of two or more thereof.
 8. A process for thepreparation of an analogue as claimed in claim 1 comprising oxidation ofsalcatonin in the presence of a suitable agent and anhydrous methanol ata temperature in the range of from -10° C. to 20° C. for up toapproximately 21/2 hours followed by purification.
 9. A process asclaimed in claim 8 wherein the suitable agent is H₂ O₂, performic acid,percloric acid, O₃, chloranil or a combination of two or more thereof.10. An analogue as claimed in claim 1 for use in the inhibition ofphysiological or pathological bone re-absorption.
 11. An analogue asclaimed in claim 1 for use as a hypocalcemic agent.
 12. An analogue asclaimed in claim 1 for administration in a daily dose of 10 I.U. to 400I.U.
 13. An analogue as claimed in claim 1 for administration in a dailydose of 100 I.U. to 200 I.U.
 14. A process for determining the contentof hydroxysalcatonins in a salcatonin preparation comprising comparingthe amount of hydroxysalcatonin in the salcatonin preparation to astandard containing a known amount of a hydroxysalcatonin analogue asclaimed in claim
 1. 15. A process as claimed in claim 14 which is achromatographic analytical test.